Bone polypeptide-1

ABSTRACT

The present invention relates to a bone polypeptide, particularly bone polypeptide-1 and nucleic acid molecules encoding the same. The present invention provides the human primary sequence of bone polypeptide-1 as well as sequences of vertebrate homologs. The invention also provides cell lines engineered to express the cDNA, antibodies to detect its translation products, and recombinant adenoviruses to deliver an expressible cDNA into a host. Bone polypeptide-1 can be utilized to treat diseases affecting renal and bone functions.

RELATED APPLICATION

This application is a 371 of PCT Application No. PCT/IB02/05778, filedDec. 20, 2002, which claims the benefit of U.S. Provisional Application60/341,224 filed Dec. 20, 2001, all documents are incorporated herein byreference.

BACKGROUND OF THE INVENTION

a) Field of the invention

The present invention relates to a bone polypeptide and active fragmentsthereof. In particular, the present invention relates to bonepolypeptide-1, active fragments thereof and nucleic acids encoding thesame.

b) Brief Description of the Prior Art

Bone is a specialized connective tissue that is constantly remodelled bythe reciprocal action of bone-forming cells (osteoblasts) andbone-resorbing cells (osteoclasts) (Manolagas, 2000). Full citations forthe references cited herein are provided before the claims. In thedeveloping organism, the skeletal system is formed either throughendochondral or intramembranous ossification (Baron, 1999). Endochondralbone formation, exemplified by the development of the long bones of thelimbs, involves replacement of a preformed cartilage template whereasintramembranous ossification, a process typical of flat bones of theskull, does not rely on an intermediate cartilaginous step. In bothcases, osteoblasts originate from the mesenchyme and initiallydifferentiate from the inner layer of the periosteum. Periostealosteoblasts deposit lamellar bone, progressively becoming enthumbed inthe matrix they deposit and finally differentiating into osteocytes.Osteocytes elaborate cytoplasmic extensions through canaliculi, therebyconstituting a network of interconnected cells within bone tissue(Aarder et al., 1994). It is thought that this network senses load onthe bones and alters bone activity according to the demands of thatload.

Osteoporosis is characterized by a loss of bone mass due to an imbalancebetween bone resorption and bone formation. This degenerative diseaseaffects 20 million women in the United States. Current treatment mainlyinvolves the inhibition of the activity of osteoclasts by inhibitorssuch as bisphosphonates (Russell, 1999). Such anti-resorptive therapiesslow down the progression of the disease but do not really help inrebuilding lost bone. Effective bone anabolics are thus needed.Unfortunately, with the possible exception of a fragment of parathyroidhormone (PTH₁₋₃₄; Neer et al., 2001), very few molecules have been shownto notably increase bone mass in osteoporotic patients. A number ofgrowth factors and related molecules are now being considered astherapeutic agents. Insulin-like growth factor 1, (IGF1) is a proteinthat displays bone-sparing activity in the ovariectomized rat, a modelof postmenopausal osteoporosis (Bagi et al., 1995). Basic fibroblastgrowth factor (bFGF) is another protein that was shown to enhance boneformation in vivo (Mayahara et al., 1993). However IGF1, as the nameimplies, is also an hypoglycemic factor and bFGF severely disruptshematopoiesis. Hence, the in vivo specificity of these growth factors isdifficult to ascertain and their clinical potential is unproven. Thereis thus a need to identify molecules that can efficiently andspecifically increase bone formation.

Regulation of bone mass by the central nervous system has recently beenreported by Ducy and colleagues (Ducy et al., 2000). These authors haveshown that the ob/ob mice lacking a functional leptin gene have a higherbone mass. Leptin is a 16 kD hormone synthesized by the adipocytes andacting on hypothalamic neurons to regulate caloric intake (Unger, 2000).When injected intracerebroventricularly, leptin caused a loss of bone.It has been hypothesized that, in addition to adipocytes, osteoblastsper se might secrete a humoral factor that regulates bone mass throughan hypothalamic relay. Thus, identification of such a factor might beuseful to devise new therapies for the treatment of osteoporosis.

The inorganic component of bone is made up of hydroxyapatite crystals.Hence, in addition to its role as a supporting mechanical structure, theskeleton is a reservoir of calcium. Bone cells play a crucial role inthe homeostasis of calcium and other ions such as phosphate. Inparticular, it has been proposed that bone cells secrete a hormone,tentatively called phosphatonin, that regulates phosphate retention bykidney tubules. It has been reported that phosphatonin levels areelevated in conditioned medium of tumor cells derived from a raredisease called oncogenic hypophosphatemia osteomalacia (Kumar, 2000).Furthermore, it has been postulated that phosphatonin is a substrate forPhex, a metallopeptidase found at the cell surface of osteoblasts andosteocytes (Frota Ruchon et al., 2000). This hypothesis is supported bythe fact that patients with X-linked hypophosphatemia harbor deleteriousmutations in the Phex coding sequence (The Hyp consortium, 1995) andpresumably have elevated levels of active phosphatonin. Low serumphosphate levels are associated with impaired bone quality.Hypophosphatemia could be remedied by injection of phosphatoninantagonists. In view of the above, it is clear that there is a need toidentify the molecular nature of phosphatonin or other molecules thatregulate phosphate metabolism. In particular, there is a tremendous needto identify bone polypeptides that may be useful as therapeutic agents.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed torecombinant bone polypeptide-1 (BP-1), proteins sharing substantialhomology to BP-1 and active fragments thereof. Bone polypeptide-1 ispolypeptide expressed in bone. Bone polypeptide 1 can be synthesizedchemically, recombinantly produced, isolated and/or purified from arecombinant host or it and it can be isolated and/or purified from itsnatural source. Sources of bone polypeptide-1 include all organismscontaining bone since bone polypeptide-1 is predominantly expressed inbone cells. Preferred sources of bone polypeptide 1 include rat, mouse,python, cow and chicken. An especially preferred source of bonepolypeptide-1 is a human.

The present invention is further directed to nucleic acids encoding bonepolypeptide-1 and active fragments thereof; a vector containing thenucleic acids and a host cell carrying the vector. The present inventionis further directed to processes to produce bone polypeptide-1 andactive fragments thereof, processes to produce cells capable ofproducing the bone polypeptide-1 and active fragments thereof and toproduce a vector containing DNA or RNA encoding bone polypeptide-1 andactive fragments thereof. The present invention is further directed tomethods for treating bone and renal diseases and methods for usingpharmacologic compositions comprising an effective amount of bonepolypeptide-1 and active fragments thereof. The invention alsoencompasses monoclonal and polyclonal antibodies specificallyrecognizing bone polypeptide-1 and active fragments thereof.

The present invention is further directed to an isolated nucleic acidencoding bone polypeptide-1 and active fragments thereof. The nucleicacid may be isolated from an animal, preferably a human. Other sourcesof the nucleic acid include rats, cows, snakes, mice and chickens.

Bone polypeptide-1 may regulate bone cell proliferation and/or bone celldifferentiation and/or osteoblast activity via an autocrine, paracrineor endocrine pathway. Active fragments of bone polypeptide-1 arefragments that have similar activity as the full length protein andtherefore may regulate bone cell proliferation and/or bone celldifferentiation and/or osteoblast activity via an autocrine, paracrineor endocrine pathway.

The present invention is further directed to nucleic acids havingsequences selected from: SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7 and homologous sequences.

The present invention is further directed to vector including a nucleicacid encoding bone polypeptide-1 and active fragments thereof. In oneformat, the vector includes an expression control sequence operablylinked to the nucleic acid. The expression control sequence may be apromoter such as a prokaryotic promoter or a eukaryotic promoter. Thevector may be present in an isolated cell such as a prokaryotic cell ora eukaryotic cell.

The present invention is further directed to a method of producing bonepolypeptide-1 or active fragments thereof by culturing a cell containingan vector containing a nucleic acid encoding bone polypeptide-1 underconditions permitting expression of the polypeptide and purifying thepolypeptide from the cell or culture medium of the cell. The cell may bea prokaryotic cell or a eukaryotic cell.

The present invention is further directed to an isolated nucleic acidthat hybridizes under high stringency conditions to a nucleic acidincluding nucleotide sequences such as SEQ ID NO: 2; SEQ ID NO: 3; SEQID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, andthe complements thereof wherein the high stringency conditions includeshybridizing in 0.15 M NaCl at 72° C. for about 15 minutes and washingthe hybridized DNA in 0.2×SSC at 65° C. for 15 minutes. The nucleic acidmay be isolated from an animal, preferably a human. Other sources of thenucleic acid include rats, cows, snakes, mice and chickens.

The nucleic acids of the invention may be cloned into an vector. Thevector may further include an expression control sequence operablylinked to the nucleic acid. The expression control sequence may be apromoter such as a prokaryotic promoter or a eukaryotic promoter.

The present invention is further directed to isolated and/or purifiedand/or recombinant bone polypeptide-1 or an active fragment thereof in apharmaceutical composition. The polypeptide may be isolated and/orpurified from a human. Alternatively, the polypeptide may berecombinantly produced from the nucleic acid encoding the humanpolypeptide. The polypeptide may also be isolated from rat, cow, snake,mouse or chicken. Alternatively, the polypeptide may be recombinantlyproduced from the nucleic acid encoding the rat, cow, snake, mouse orchicken polypeptide.

The present invention is further directed to polypeptides having aminoacid sequences selected from of SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO:11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14, SEQ ID NO: 20, SEQ IDNO: 21 SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

The present invention is further directed to an expression vectorcontaining a coding sequence the sequence of which encodes bonepolypeptide-1 or an active fragment thereof, the vector being suitablefor genetic therapy.

The present invention is further directed to a method to deliver anucleic acid, the sequence of which encodes bone polypeptide-1 or afragment thereof, into a host for therapy. In one format of the methodof the invention, the delivery is by adenovirus.

The present invention is further directed to a method of treating a bonedisorder or osteoporosis comprising administering an effective amount ofbone polypeptide-1 or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, B show the results of a Northern analysis comparing theexpression of a mouse cDNA encoding bone polypeptide-1 (BP-1) in avariety of embryonic and adult mouse tissues. A. Five micrograms oftotal RNA was loaded in lanes 1 to 16. 1, brain e11.5; 2, adult brain;3, gonads e14.5; 4, adult testis; 5, heart e12.5; 6, adult heart; 7,intestine e14.5; 8, adult intestine; 9, kidney e13.5; 10, adult kidney;11, liver e11.5; 12, adult liver; 13, lung e12.5; 14, adult lung; 15,spleen e15.5; 16, adult spleen. B. One and a half microgram of polyA(+)RNA was loaded in lanes 21 to 23. 21, calvaria e15.5; 22, adult kidney;23, adult liver. Bars indicate migration of 28S and 18S ribosomal RNA.

FIG. 2 shows the nucleotide sequence of a full length mouse cDNAencoding bone protein-1 (SEQ ID NO:1). The open reading frame is writtenin capital letters, the polyadenylation signal is in bold characters,the sequence corresponding to the original ca155-cap1-c2-neg306 clone isunderlined.

FIGS. 3A-F show brightfield (A,D) and darkfield (B,C,E,F) images ofsections through the limb of a mouse embryo at 16 days of gestation(A-C) or through the head of a mouse embryo at 13 days of gestation(D-F) hybridized with a probe complementary to a novel cDNA encodingbone polypeptide-1 (B,E) or to Cbfa1, a transcription factor expressedmainly in the osteoblast lineage (C,F).

FIG. 4 shows an alignment of the coding sequences from human (Homosapiens) (SEQ ID NO:2), mouse (Mus musculus) (SEQ ID NO:3), rat (Rattusnorvegicus) (SEQ ID NO:4), bovine (Bos taurus) (SEQ ID NO:5), chicken(Gallus gallus) (SEQ ID NO:6), and python (Python molurus bivittatus)(SEQ ID NO: 7) BP-1 cDNAs. The consensus DNA sequence for BP-1 isprovided as SEQ ID NO: 8.

FIG. 5A shows an alignment of the primary sequences of BP deduced fromcDNAs isolated from tissues of the human (Homo sapiens) (SEQ ID NO:9),mouse (Mus musculus) (SEQ ID NO:10), rat (Rattus norvegicus) (SEQ IDNO:11), bovine (Bos taurus) (SEQ ID NO:12), chicken (Gallus gallus) (SEQID NO:13) and python (Python molurus biviffatus) (SEQ ID NO:14). Theconsensus polypeptide sequence for BP-1 is provided as SEQ ID NO: 15.Sequence of the putative signal peptides are underlined. Putativeprocessing sites are boxed.

FIG. 5B shows an alignment of a region of homology between BP-1 andmembers of the natriuretic peptides family. Numbering of amino acids isaccording to the full length precursor protein (SEQ ID NO:9 for BP-1).The human sequence is SEQ ID NO: 16. The rat atrial natriuretic factorprotein region is SEQ ID NO: 17. The rat brain natriuretic factorprotein region is SEQ ID NO: 18. The rat C-type natriuretic factorprotein region is SEQ ID NO: 19.

FIG. 6A depicts the full length protein derived from the human BP-1 cDNA(SEQ ID NO: 2) and is presented as SEQ ID NO: 9. The signal peptide andcorresponding signal peptidase cleavage site are shown. Also shown arethe conserved dibasic cleavage sites, the putative products generated byprocessing of the protein (PROT 1 (SEQ ID NO: 20), PEPT 1 (SEQ ID NO:21), PEPT 2A (SEQ ID NO: 22), PEPT 3A (SEQ ID NO: 23), PEPT 2B (SEQ IDNO: 24), PEPT 3B (SEQ ID NO: 25)), and the position of syntheticpeptides used to raise antibodies (AG1 (SEQ ID NO: 26), AG2 (SEQ ID NO:27), AG3 (SEQ ID NO: 28)). The residues at the N and C termini ofpeptides are indicated and numbered according to the initiatormethionine of SEQ ID NO:9.

FIG. 6B shows a picture of Western analysis on cell extracts (lanes 620)or precipitated medium (lanes 621) from cells transfected with a vectorexpressing wild type BP-1 mouse cDNA (lanes 620, 621, 622), or a form ofBP-1 mouse cDNA where one of the dibasic cleavage site was mutated(lanes 623). Migration of molecular weight markers is indicated.

FIG. 7A depicts an expression vector for BP-1 (plasmid GD46) that can beused to generate cell lines secreting the BP-1 polypeptide or fragmentsthereof in the culture medium. The vector comprises components of atranscription unit for mouse BP-1 (700, 701, 702) as well as atranscription unit to confer resistance to puromycin (703).

FIG. 7B shows the standard curve of an enzyme-linked immunoadsorbentassay (ELISA) performed on increasing quantities of synthetic peptidederived from the C terminal region of BP-1.

FIG. 8A shows the levels of BP-1 immunoreactivity in fractions collectedfrom a cation-exchange chromatography column after passage ofconditioned medium from 293-GD46-7 cells overexpressing the mouse BP-1coding sequence.

FIG. 8B is a picture of a silver-stained polyacrylamide gel showing theproteins found in the conditioned medium from 293-GD46-7 cells (lane810), in the flow through from a cation-exchange chromatography column(lane 811) and in BP-1-immunoreactive fractions from the same column(lane 812). Arrow 813 points to a BP-1 product. Arrowhead 814 points toaprotinin, a contaminating protease inhibitor included in theconditioned medium before the chromatographic step.

FIG. 8C shows the result of a Western analysis performed on conditionedmedium from 293-GD46-7 cells (lane 820), on the flow through from acation-exchange chromatography column (lane 821) and onBP-1-immunoreactive fractions from the same column (lane 822).

FIG. 9A depicts a vector (plasmid GD28b) that can be used to generaterecombinant adenoviral particles expressing BP-1 coding sequence. Thevector comprises a transcription unit for mouse BP-1 (901) flanked bytwo regions of the adenovirus serotype 5 genome (902, 903).

FIG. 9B shows the result of an ELISA to detect BP-1 products in themedium of primary cultures of rat osteoblasts infected with recombinantadenoviral particles at day 11 of culture.

FIG. 10 shows the result of a Northern analysis to detect osteocalcin(OCN) in total RNA extracted from primary cultures of osteoblaststreated chronically with control medium (1001) or medium containing BP-1products (1002). The levels of a control messenger (GAPDH) are alsoshown.

DETAILED DESCRIPTION OF THE INVENTION

A) Definitions

Throughout the text, the word “kilobase” is generally abbreviated as“kb”, the words “deoxyribonucleic acid” as “DNA”, the words “ribonucleicacid” as “RNA”, the words “complementary DNA” as “cDNA”, the words“polymerase chain reaction” as “PCR”, the words “expressed sequencedtag” as “EST”, and the words “reverse transcription” as “RT”. Nucleotidesequences are written in the 5′ to 3′ orientation unless statedotherwise. Amino acid sequences are written from the N terminus unlessstated otherwise.

In order to provide an even clearer and more consistent understanding ofthe specification and the claims, including the scope given herein tosuch terms, the following definitions are provided:

Exogenous nucleic acid: A nucleic acid (such as cDNA, cDNA fragments,genomic DNA fragments, mRNA fragments, antisense RNA, oligonucleotide)which is not naturally part of another nucleic acid molecule. The“exogenous nucleic acid” may be from any organism, purely synthetic, orany combination thereof.

Expressed sequenced tag: sequence information on a small nucleic acid(typically 200-500 bp) derived from a gene transcript.

Hormone: a molecule, generally polypeptidic in nature, secretedextracellularly by one cell type to modulate the activity of targetcells.

Hormone precursor. Refers to a secreted protein that is processed in thesecretory pathway such that one or more fragments are releasedextracellularly. Fragments can be further modified (e.g. amidation atthe C terminus) before or after release in the extracellular space.

Host: A cell, tissue, organ or organism capable of providing cellularcomponents for allowing the expression of an exogenous nucleic acid. Theexogenous nucleic acid maybe cloned into a vector. This term is intendedto also include hosts which have been modified in order to accomplishthese functions. Bacteria, fungi, animal (cells, tissues, or organisms)and plant (cells, tissues, or organisms) are examples of a host.

Insertion: The process by which a nucleic acid is introduced intoanother nucleic acid. Methods for inserting a nucleic acid into anothernormally requires the use of restriction enzymes and such methods ofinsertion are well known in the art.

In silico: using computer and bioinformatics software and hardware.

Nucleic acid: Any DNA, RNA sequence or molecule having one nucleotide ormore, including nucleotide sequences encoding a complete gene. The termis intended to encompass all nucleic acids whether occurring naturallyor non-naturally in a particular cell, tissue or organism. This includesDNA and fragments thereof, RNA and fragments thereof, cDNAs andfragments thereof, expressed sequence tags, artificial sequencesincluding randomized artificial sequences.

Open reading frame (“ORF”). The portion of a cDNA that is translatedinto a protein. Typically, an open reading frame starts with aninitiator ATG codon and ends with a termination codon (TAA, TAG or TGA).

Protein products: refers to the various peptides or polypeptidesgenerated after translation of a single mRNA. Polypeptides refer toamino acids linked to form a peptide or a full length protein.

Recombinant: The term “recombinant” in association with “vector” refersto a vector which has been modified to contain a non-native exogenousnucleic acid.

Secreted protein: any protein that enters the cellular secretorypathway. Typically, secreted proteins are synthesized bearing a signalpeptide which is removed shortly after its synthesis.

Signal peptide: a peptide capable of directing a nascent protein to thecell secretory pathway. It is generally accepted that a signal peptideis composed of an initiating methionine, a highly hydrophobic stretch,typically 10 to 15 residues long, and a signal peptidase cleavage site.

Transcription unit: As used herein, a “transcription unit” refers to anucleic acid which comprises an enhancer sequence, a promoter sequence,and a transcription termination sequence, all operably linked together.Preferably, the enhancer and promoter sequences are constitutivelyactive in various hosts. Enhancer and promoter sequences can be derivedfor example from the cytomegalovirus (CMV) immediate-early genes or fromthe Rous sarcoma virus (RSV) long terminal repeat. Preferably, thetranscription unit is comprised within a vector.

Transfection: the process of introducing nucleic acids in eukaryoticcells by any means such as electroporation, lipofection, precipitateuptake, micro-injection. A cell having incorporated an exogenous nucleicacid is said to be transfected.

Vector: A RNA or DNA molecule which can be used to transfer an RNA orDNA segment from one organism to another. Vectors are particularlyuseful for manipulating genetic constructs and different vectors mayhave properties particularly appropriate to express protein(s) in arecipient during cloning procedures and may comprise differentselectable markers. Bacterial plasmids are commonly used vectors.

B) General Overview of the Invention

The invention is based on isolated nucleic acids encoding bonepolypeptide-1. The invention encompasses isolated or substantiallypurified nucleic acid or protein compositions. In the context of thepresent invention, an “isolated” or “substantially purified” DNAmolecule or an “isolated” or “substantially purified” polypeptide is aDNA molecule or polypeptide that, by the hand of man, exists apart fromits native environment and is therefore not a product of nature. Anisolated DNA molecule or polypeptide may exist in a purified form or mayexist in a non-native environment such as, for example, a transgenichost cell. An isolated or purified DNA or polypeptide may be synthesizedchemically, may be produced using recombinant DNA techniques and thenisolated or purified or may be isolated or purified from its naturalhost. An “isolated” or “substantially purified” nucleic acid molecule orprotein, or biologically active portion thereof, is substantially freeof other cellular material, or culture medium when produced byrecombinant techniques and, in some circumstances, further purifed, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Preferably, an “isolated” nucleic acid is freeof sequences (preferably protein encoding sequences) that naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. A protein that is substantially free ofcellular material includes preparations of protein or polypeptide havingless than about 30%, 20%, 10%, 5%, (by dry weight) of contaminatingprotein. When the protein of the invention, or biologically activeportion thereof, is recombinantly produced, preferably culture mediumrepresents less than about 30%, 20%, 10%, or 5% (by dry weight) ofchemical precursors or non-protein of interest chemicals.

i) Cloning of a cDNA Fragment Encoding Bone Polypetide-1

We have previously developed a screening system that allows the rapididentification of nucleic acids encoding signal peptides from complexlibraries of cDNA fragments (International patent application No.PCT/CA01/01169; U.S. patent application Ser. No. 09/641,931). A libraryenriched in 5′ fragments of cDNAs derived from developing calvaria wasobtained using the so-called “oligo-capping” method (Maruyama andSugano, 1994). Briefly, the top halves of skulls of e15.5 mouse embryoswere dissected free from nervous or skin tissue. Total RNA was extractedusing the guanidium/phenol method and mRNA was isolated using OligotexmRNA kit (Qiagen, Mississauga, Canada). A synthetic oligoribonucleotide(RNA30-1; 5′-agcaucgagucggccuuguuggccuacugg-3′) SEQ ID NO:29 wasspecifically ligated to the 5′ extremities of mRNAs whose CAP moiety hadbeen converted to a free phosphate group by the action of tobacco acidpyrophosphatase. The first strand of corresponding cDNA was synthesizedusing a primer-linker comprising a random sequence of 9 nucleotides(36-2V; 5′-gagagagagagagcgactcggatccannnnnnnnnc-3′) SEQ ID NO 30. Theresulting first strand cDNA was amplified by 30 cycles of PCR usingprimers complementary to both extremities (18-114V;5′-agcatcgagtcggccttg-3′) SEQ ID NO: 31 and (18-99V;5′-gagagcgactcggatcca-3′) SEQ ID NO: 32. Amplicons were purified andsubjected to a further 10 cycles of semi-nested PCR using primers 18-99Vand 32-4V (5′-ggacgagcggccgcgccttgttggcctactgg-3′) SEQ ID NO:33. PCRproducts were digested with NotI and BamHI, size-selected byelectrophoresis (500-1200 bp) and directionally cloned in anexpression/screening vector digested with NotI and BamHI. The librarynumbered approximately 3,000,000 primary clones. Sequencing of randomlychosen inserts revealed that the average insert size was 575 bp and that60% of inserts corresponded to 5′ fragments of cDNAs. The library wasexpression screened in BHK-21 fibroblasts. Selected fragments wereamplified, cloned in the pBluescript KS II+ vector and sequenced using aCEQ2000™ automated sequencer.

One of the retrieved fragments (ca155-cap1-c2-neg306, 433 bp in length)encoded a putative translation product of 124 residues containing asignal peptide. Interestingly, this translation product harboredsequences reminiscent of dibasic cleavage sites found in peptide hormoneprecursor (KKKR (SEQ ID NO: 132) at position 76-79 and KKR at position110-112). This observation prompted us to determine the expressionprofile of the corresponding gene by Northern analysis of RNA extractedfrom 52 different tissues and cell lines. No signal was detected afteran exposure of 7 days; a representative autoradiograph is shown in FIG.1A. In order to verify that the retrieved fragment derived from acellular transcript, Northern analysis was performed with 1.5 μg ofpolyA+RNA extracted from e15.5 mouse calvaria, adult liver and adultkidney (FIG. 1B). A clear signal (arrow; 25), corresponding to a mRNA ofapproximately 1.3 kb, was specifically detected in the embryoniccalvaria. Taken together, these results indicate thatca155-cap1-c2-neg306 is derived from a mRNA that encodes a bone proteinthat is specifically expressed in bone tissue.

ii) Results from Database Mining; Cloning of Full Length Mouse cDNAContaining c155-cap1-c2-neg306

Because of the interesting features of clone ca155-cap1-c2-neg306, wecarried out extensive database searches to compare its sequence withthose deposited in the public domain. We used the standard Blastn tool(v2.2.4, Aug. 26, 2002) with the following parameter set: expect valueof 10, low complexity filter. There was only one significant match to anentry (accession XM_(—)155941.1) in the non redundant (nr) set of theGenbank™ database. This clone (designated LOC239790) encodes a putativeprotein whose N terminal sequence is identical to that ofca155-cap1-c2-neg306 but diverges at residues GIn¹⁰². Searches in dbESTrevealed that ca155-cap1-c2-neg306 shows high homology to a bovine EST(77.5% homology, accession number BF045261), to an uncharacterized mouseEST (accession number BB638598.1), to 3 human ESTs from metastaticchondrosarcoma (accession numbers BQ021661, BQ001512, BQ000995) and thatthe last 102 nucleotides of ca155-cap1-c2-neg306 shows high homology(85.8%) to the last 105 nucleotides of a rat EST (accession numberAl178209). It should be noted that a valid ORF can not be deduced fromthe sequences of either mouse, rat or human ESTs. The 526 bp bovine ESTcontained an ORF of 132 residues. In silico assembly ofca155-cap1-c2-neg306 and the rat EST yielded a 0.99 kb chimericsequence, roughly corresponding to the size of the expected full lengthtranscript minus polyA tail (see Northern analysis, FIG. 1B).

Since we had initially cloned the 5′ end of ca155-cap1-c2-neg306, weused a modified 3′ RACE strategy to obtain a full length cDNA (seeMaterials and Methods in the Examples section). Sequences from at leastfive different clones were aligned together with ca155-cap1-c2-neg306 toreconstitute the full length consensus cDNA sequence shown on FIG. 2.The 1280 bp cDNA (SEQ ID NO:1) contains an ORF of 393 bp (uppercase)flanked by 61 bp and 811 bp of untranslated sequences at the 5′ and 3′ends, respectively (lowercase). A polyadenylation signal is found 14 bpupstream of a polyA stretch. The putative initiator ATG codon is foundin an adequate Kozak context (AAGATGC, SEQ ID NO: 25).

iii) Expression Profiling on Histological Sections of Mouse Embryos

To determine which bone cells express the cDNA from whichca155-cap1-c2-neg306 derives, in situ hybridization was performed onsections of e13.5 and e16.5 mouse embryos using standard procedures.Consecutive sections were hybridized with a probe partiallycomplementary to Cbfa1 transcripts. Cbfa1 is a transcription factorexpressed in cells of the osteoblast lineage and whose activity isessential for proper development of the skeleton (Ducy, 2000). As shownin FIG. 3B, the ca155-cap1-c2-neg306 probe specifically labels cellssurrounding the bone collar in e16.5 limbs. These cells are located inthe periosteum and are cells of the osteoblast lineage. This is revealedby the fact that they express Cbfa1 (FIG. 3C). At e13.5, expression ofca155-cap1-c2-neg306 is seen in a few cells in the vicinity ofcartilaginous formations in the skull (FIG. 3E). These cells are alsolabeled by the Cbfa1 probe (FIG. 3F). Thus, the cDNA from whichca155-cap1-c2-neg306 derives is expressed in cells of the osteoblastlineage.

iv) Cloning of Vertebrate Homologs

Because of its features, the cDNA from which ca155-cap1-c2-neg306derives will be referred hereafter as mouse BP-1, mouse bonepolypeptide-1. As discussed in more detail below, various homologs,including at least one human homolog to BP-1 exist. Bone polypeptide-1,as defined herein, refers to a polypeptide expressed in bone. Asdiscussed in more detail below, bone polypeptide-1 may regulate bonecell proliferation and/or bone cell differentiation and/or osteoblastactivity via an autocrine, paracrine or endocrine pathway.

BP-1 DNA Sequences

The BP-1 DNA used in any embodiment of this invention can be BP-1 cDNAobtained as described herein, or alternatively, can be anyoligonucleotide sequence having all or a portion of a sequencerepresented herein, or their functional equivalents. Sucholigodeoxynucleotide sequences can be produced chemically ormechanically, using known techniques.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length BP-1 cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, 1988; the local homology algorithm of Smith et al.1981; the homology alignment algorithm of Needleman and Wunsch 1970; thesearch-for-similarity-method of Pearson and Lipman 1988; the algorithmof Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.1988; Higgins et al. 1989; Corpet et al. 1988; Huang et al. 1992; andPearson et al. 1994. The ALIGN program is based on the algorithm ofMyers and Miller, supra. The BLAST programs of Altschul et al., 1990,are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through theweb site for National Center for Biotechnology Information (NCBI). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,1990). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always>0) and N (penalty scorefor mismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when the cumulative alignment scorefalls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. 1997.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al., supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.BLASTN for nucleotide sequences, BLASTX for proteins) can be used. TheBLASTN program (for nucleotide sequences) uses as defaults a wordlength(W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See theNCBI web site. Alignment may also be performed manually by inspection.

For purposes of the present invention, comparison of BP-1 nucleotidesequences for determination of percent sequence identity to the BP-1sequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo BP-1 nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a BP-1 polynucleotide comprises a sequence that has at least 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%,or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequenceidentity, compared to a reference sequence using one of the alignmentprograms described using standard parameters. One of skill in the artwill recognize that these values can be appropriately adjusted todetermine corresponding identity of proteins encoded by two nucleotidesequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like. Substantialidentity of amino acid sequences for these purposes normally meanssequence identity of at least 70%, more preferably at least 80%, 90%,and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a BP-1 peptideindicates that a peptide comprises a sequence with at least 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%,or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity tothe reference sequence over a specified comparison window. Preferably,optimal alignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970). An indication that two peptide sequencesare substantially identical is that one peptide is immunologicallyreactive with antibodies raised against the second peptide. Thus, apeptide is substantially identical to a second peptide, for example,where the two peptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two BP-1 nucleic acid sequencesare substantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridization are sequence dependent, andare different under different environmental parameters. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, 1984; T_(m) 81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. T_(m) is reduced by about1° C. for each 1% of mismatching; thus, T_(m), hybridization, and/orwash conditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point I forthe specific sequence and its complement at a defined ionic strength andpH. However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point I;moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the thermal melting point I; lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point I. Using theequation, hybridization and wash compositions, and desired T, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, 1993. Generally, highly stringent hybridization and washconditions are selected to be about 5° C. lower than the thermal meltingpoint T_(m) for the specific sequence at a defined ionic strength andpH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M, more preferably about 0.01 to 1.0 M, Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. and at least about 60° C. for long robes (e.g., >50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone orthologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

Protein Sequences

The present invention includes peptides which are derivable from BP-1. Apeptide is said to be “derivable from a naturally occurring BP-1 aminoacid sequence” if it can be obtained by fragmenting a naturallyoccurring sequence, or if it can be synthesized based upon a knowledgeof the sequence of the naturally occurring BP-1 amino acid sequence orof the genetic material (DNA or RNA) which encodes this sequence.

Included within the scope of the present invention are those moleculeswhich can be said to be “derivatives” or “active fragments” of BP-1referred herein as “BP-1 products”. Such a “derivative” or “activefragment” has one or more of the following characteristics: (1) itshares substantial homology with BP-1 or a similarly sized fragment ofBP-1; (2) it is capable of functioning as a bone hormone and (3) usingat least one of the assays provided herein, the derivative has either(i) a bone hormone activity, or, (ii) an activity on the kidney. Bonehormone activity refers to an ability to regulate bone cellproliferation and/or bone cell differentiation and/or of osteoblastactivity, via an autocrine, paracrine or endocrine pathways.

A derivative of BP-1 is said to share “substantial homology” with BP-1if the amino acid sequences of the derivative is at least 60%, and morepreferably at least 80%, and most preferably at least 90%, the same asthat of BP-1.

The derivatives of the present invention include BP-1 fragments which,in addition to containing a sequence that is substantially homologous tothat of a naturally occurring BP-1 peptide may contain one or moreadditional amino acids at their amino and/or their carboxy termini.Thus, the invention pertains to polypeptide fragments of BP-1 that maycontain one or more amino acids that may not be present in a naturallyoccurring BP-1 sequence. The additional amino acids may be D-amino acidsor L-amino acids or combinations thereof. Furthermore, the additionalamino acids may be naturally occurring amino acids or non-naturallyoccurring amino acids such as L-tert-leucine; L-homophenylalanine;D-homophenylalanine; D-methionine; Halogenated D and L-phenylalanines,tyrosines, and tryptophans; D-2-aminopimelic acid and L-2-aminopimelicacid

The invention also includes BP-1 fragments which, although containing asequence that is substantially homologous to that of a naturallyoccurring BP-1 peptide may lack one or more additional amino acids attheir amino and/or their carboxy termini that are naturally found on aBP-1 peptide. Thus, the invention pertains to polypeptide fragments ofBP-1 that may lack one or more amino acids that are normally present ina naturally occurring BP-1.

The invention also encompasses the obvious or trivial variants of theabove-described fragments which have inconsequential amino acidsubstitutions (and thus have amino acid sequences which differ from thatof the natural sequence) provided that such variants have a bone hormoneactivity which is substantially identical to that of the above-describedBP-1 derivatives. Examples of obvious or trivial substitutions includethe substitution of one basic residue for another (i.e. Arg for Lys),the substitution of one hydrophobic residue for another (i.e. Leu forIle), or the substitution of one aromatic residue for another (i.e. Phefor Tyr), etc.

Bone Polypeptide-1 Homologs

A cDNA encoding BP-1, or a portion thereof can be used to identifysimilar sequences in other vertebrate species and thus, to identify or“pull out” sequences which have sufficient homology to hybridize to BP-1cDNA or mRNA or a portion thereof under conditions of low stringency.Those sequences which have sufficient homology (generally greater than40%) can be selected for further assessment using the method describedherein. Alternatively, high stringency conditions can be used asdiscussed above. In this manner, DNA of the present invention can beused to identify, in other species, sequences encoding polypeptideshaving amino acid sequences similar to that of BP-1 and thus to identifybone polypeptides in other species. Thus, the present invention includesnot only BP-1, but also related proteins encoded by DNA whichhybridizes, preferably under high stringency conditions, to DNA of thepresent invention.

BLAST searches of the mouse BP-1 sequence using the Ensembl web site(containing the sequence of the human genome, v8.30a.1, August 2002data) revealed that a human homolog of BP-1 might exist. Indeed,homologous sequences are found on human chromosome 3q28 in contigsAC019234.5.48926.64909 (nt 12659-12937, 75% homology),AC019234.5.130697.209258 (nt 75405-75529, 79% homology),AC019234.5.89412.130596 (nt 39498-39560, 92% homology). The Genescanalgorithm predicts 4 exons but those are disordered and not annotated aspart of a single gene and, furthermore, the predicted translationproduct do not match the primary sequence of BP-1, presumably becauseGenescan has not identified the correct ORF. Thus, the ORF predictedfrom the BP-1 cDNA (FIG. 2) can not be determined solely by in silicoanalysis of the sequence of the human genome. Therefore, in order toclone a human homolog of BP-1, we performed RT-PCR on human bone marrowmRNA using two primers encompassing the putative initiator andtermination codons found in the human genome sequence. In addition, therat homolog of BP was cloned by low-stringency RT-PCR using one primerencompassing the putative initiator codon and another encompassing aregion in the 3′ untranslated region of the mouse cDNA. To identifyevolutionarily conserved domains within the BP-1 protein, we obtainedsequence information on BP-1 from non mammalian species. The cDNAencoding chicken (Gallus gallus) BP-1 was retrieved by ‘BLASTing’ thehuman ORF against the BBSRC Chicken EST Project database (Blastn tool,all tissues, matrix BLOSUM62, expectation 10⁻²). A portion of the cDNAencoding snake (Python molurus bivittatus) BP-1 was cloned by RT-PCRusing degenerate oligonucleotide primers and starting from RNA extractedfrom the vertebrae of a young python (See Materials and Methods in theExamples section.) FIG. 4 shows the alignment of the nucleic acidsequences encoding human, bovine, mouse, rat, chicken, and python BP-1.Allelic variations of the nucleic acid sequences described herein arealso encompassed in the invention.

FIG. 5A shows the alignment of the ORFs derived from the BP-1 cDNAs ofmouse, human, rat, cow, chicken. The partial ORF deduced from the pythonsequence is also aligned. The presence of a cleavable signal peptide aswell as the position of the putative processing sites are conservedacross species (respectively underlined and boxed in FIG. 5A). In silicoanalysis indicates that a) the region of the protein that lies betweenthe 2 dibasic cleavage sites (residues 83-112 in the human sequence) iswell conserved (overall identity of 60%, overall similarity of 76%); b)the C terminal region of the protein (residues 116-133 in the humansequence) is also well conserved (human and mouse share 94.4% similarityand identity; human and chicken share 83.3% similarity and 61.1%identity). The observation that the dibasic cleavage sites are found invarious species suggests that BP-1 is a prohormone precursor conservedin terrestrial vertebrates. Furthermore, analysis of the conserveddomains suggests that the bioactive protein products derive from the Cterminal half of BP-1.

In silico analysis indicates that full length human BP-1 has thefollowing feature: it has a molecular weight of 14,832 g/mol and anisoelectric point of 9.62; it contains neither N-glycosylation sites nordisulfide bridges. Despite extensive searches in public databases (e.g.InterPro, ProDom, STN DGENE), BP-1 shows no significant homology to anyknown protein, protein motif or protein domain. We have found howeverthat residues 116-124 of BP-1 (numbering from the human sequence) showsome homology to members of the natriuretic peptides family. FIG. 5Bshows the alignment of the homologous regions between BP-1 andnatriuretic peptides. The 2 cysteine residues conserved in allnatriuretic peptides form an intramolecular disulfide bridge essentialfor their bioactivity (Inagami et al., 1985). Cysteine residues involvedin cyclization are not found in BP-1. Interestingly however, it has beenreported that a synthetic ‘linear’ analog of the atrial natriureticpeptide could displace the binding of atrial natriuretic factor to itsreceptor but failed to activate synthesis of second messengers (Olins etal., 1988). Thus, it is possible that a BP-1 protein product could berelated both biochemically and functionally to the natriuretic peptides.

v) Production of Antibodies Against Portions of BP-1.

In order to detect the BP-1 protein products, antibodies were raisedagainst synthetic peptides derived from 3 regions of the full lengthprotein AG-1: (DELVSLENDVIETK (SEQ ID NO:26), AG-2: (RLSAGSVDHKGKQR) SEQID NO: 27, and AG-3: (MDRIGRNRLSNSRG) SEQ ID NO: 28). The chosen regionshave a high antigenicity index as predicted by the algorithm of Hopp andWoods (Hopp and Woods, 1981). The positions of the antigenic peptidesare indicated on the model of human BP-1 shown on FIG. 6A. To increaseimmunogenicity, antigenic peptides comprise a N- or C-terminal cysteineto allow covalent coupling to a carrier protein (e.g. keyhole limpethemocyanin, bovine serum albumin). The peptide/carrier complex areinjected subcutaneously into animals (e.g. rabbits) and antisera areobtained using standard protocols. (See Materials and Methods inExamples section.) Affinity chromatography was used to purifiy thefraction of immunoglobulins specific to the peptide used to elicit animmune response.

Both monoclonal and polyclonal antibodies are included within the scopeof this invention as they can be produced by well established proceduresknown to those of skill in the art. Additionally, any secondaryantibodies, either monoclonal or polyclonal, directed to the firstantibodies would also be included within the scope of this invention.

BP-1 is Secreted Extracellularly and Can be Processed

To begin assessing the biochemical and biological properties of BP-1, acDNA encoding the mouse protein was inserted into a mammalian expressionvector and this vector was transfected in HEK293A cells.Immunofluorescence analysis after cell permeabilization showed that BP-1is mainly localized to the Golgi apparatus and in small cytoplasmicvesicles of transfected cells. No signal was detected at the cellsurface. Western analysis of cell lysates and culture medium usingantibodies raised against the C terminal region of human BP-1 (AG3) (SEQID NO: 28) revealed that the bulk of the protein products is secreted inthe culture medium (FIG. 6B, compare lanes 620 and 621). The majorsecreted form has an apparent molecular weight of 13 kD, presumablycorresponding to the BP-1 precursor after removal of the signal peptide.Upon longer exposure (FIG. 6B, lane 622), a smaller product with anapparent molecular weight of ˜6 kD is reproducibly found in the culturemedium of transfected HEK293A cells. This ˜6 kD band is absent whencells are transfected with a vector expressing a form of the mouse cDNAwhere the dibasic cleavage site at position 76-79 is mutated (KKKR (SEQID NO: 132)->AS, FIG. 6B, lane 623).

Given these results, we predict that BP-1 is processed according to thefollowing scheme. After translocation in the endoplasmic reticulum, thesignal peptide is cleaved and the protein (PROT 1, SEQ ID NO: 20) may befurther processed in the secretory pathway or extracellular space toyield bioactive products. Candidate processing enzymes include furin(Denault and Leduc, 1996; GenBank™ PID accession number g31478), arelated convertase or, as discussed below, corin (GenBank™ PID accessionnumber g5729989). Our results indicate that the BP-1 precursor may becleaved at arginine⁸² (numbering according to human sequence) in anheterologous expression system (e.g. HEK293A fibroblasts) to generatePEPT 1 (SEQ ID NO: 21). Additional processing may occur C terminal toarginine¹¹⁵ at a typical dibasic site to generate PEPT 2A (SEQ ID NO:22) and PEPT 3A (SEQ ID NO: 23). Alternative processing may occur at abasic residue (lysine¹⁰⁴) upstream of the second dibasic site by anatypical convertase, similar to what has been reported for theprocessing of a natriuretic peptide precursor by the corin protease (Yanet al., 2000; Yan et al., 1999). This would generate PEPT 2B (SEQ ID NO:24) and PEPT 3B (SEQ ID NO: 25). Interestingly in this latter case, bothcleavage products of BP-1 end with a glycine residue. This observationraises the possibility that BP-1 products are amidated bypeptidylglycine alpha-amidating monooxygenase (GenBank™ accession numberno. BAC22594), an enzyme whose activity has been detected in mousecalvaria, a tissue that produces BP-1 (Bimbaum et al., 1989). FIG. 6Aillustrates the processing scenario for human BP-1 and depicts itsputative protein products.

vi) Methods of Production of Recombinant or Synthetic BP-1 ProteinProducts.

The present invention provides expression vectors and host cellstransformed to express the nucleic acid sequences encoding BP-1 of theinvention. Expression vectors of the invention comprise a nucleic acidsequence coding for at least one BP-1, or at least one antigenicfragment thereof, or derivative or homologue thereof, or the functionalequivalent of such nucleic acid sequence. Nucleic acid sequences codingfor BP-1, or at least one fragment thereof may be expressed inprokaryotic or eukaryotic host cells. Suitable host cells includebacterial cells such as E. coli, insect cells, yeast, or mammalian cellssuch as Chinese hamster ovary cells (CHO). Suitable expression vectors,promoters, enhancers, and other expression control elements may be foundin Sambrook et al. Molecular Cloning: A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989.

Whatever the producing host, the BP-1 portion of the transcription unitmay be engineered to increase or control the stability ortranslatability of the expressed mRNA, e.g. through the insertion ofexogenous cis-acting nucleic acid sequences, the replacement of poorlytranslated codons, the removal of untranslated sequences and/or theengineering of a proper context for the initiation of translation(Kozak, 1986). The BP-1 cDNA may also be engineered to increase theyield of secreted products, typically by replacing its signal peptidecoding sequence by an exogenous nucleic acid encoding a more activesignal peptide in the chosen host. Furthermore, minor modifications orvariations may be introduced in the BP-1 coding sequence to enhance thestability and/or the activity of BP-1 product(s). These modificationsmay be made by site-directed mutagenesis or may be the result ofspontaneous mutations.

Vertebrate Cell Lines

An aspect of this invention relates to modified cell lines andtransgenic organisms used to produce BP-1 protein products. According toa preferred embodiment, there is provided an eukaryotic cell line havingincorporated in its genome a DNA segment comprising a BP-1 codingsequence operably linked within a transcription unit. Enhancer andpromoter sequences driving robust expression in a wide variety of cellsare generally preferred. These include but are not limited to sequencesderived from cytomegalovirus immediate-early genes (CMV; e.g. GenBank™accession no. X03922) and Rous sarcoma virus long terminal repeat (RSV;e.g. GenBank™ accession no M83237) as well as sequences derived fromwidely expressed cellular genes such as chicken β-actin, humanelongation factor 1α, phosphoglycerate kinase, and metallothionein.Signals for the termination and polyadenylation of transcripts are wellknown in the art. Examples include part of the 3′ untranslated region ofthe bovine growth hormone gene or of the SV40 virus.

Methods to incorporate DNA segments into the genome of a cell are wellknown in the art. According to a preferred embodiment of the invention,the transcription unit is inserted into a plasmid containing a geneconferring resistance to a selective agent (e.g.puromycin-N-acetyltransferase conferring resistance to puromycin). Theresulting construct is electroporated into cells using standardprotocols. Transfection by lipofection, calcium phosphate precipitate,and micro-injection are other techniques available to introduce nucleicacids into eukaryotic cells. Selection is applied on the pool oftransfected cells. Surviving and growing cells are thought to haveincorporated the plasmid and are cloned. Individual clones are analyzedfor the production of BP-1 protein products in the culture medium.Typically, the levels of BP-1 products are determined by Westernanalysis or ELISA. Preferred cellular hosts include, but are not limitedto, human embryonic kidney 293 cells (HEK293; American Type CultureCollection no. CRL-1573) and chinese hamster ovary cells (CHO; AmericanType Culture Collection no. CCL-61). It is known in the prior art how tocultivate large quantities of these cells in order to obtain largeamounts of recombinant proteins. It is understood that a BP-1-producingcell line can be further engineered to express a convertase involved inBP-1 processing, thereby allowing the release of bioactive BP-1 productsinto the culture medium.

Transgenic Organisms

Alternatively, the transcription unit comprising the BP-1 codingsequence can be inserted into a fertilized egg (e.g. of a mouse), whichis re-implanted into a pseudo-pregnant mother. DNA extracted fromresulting organisms (e.g. embryos, pups or adults) is analyzed bySouthern blotting to determine whether the organism is transgenic.Positive animals are bred and used to produce large quantities of BP-1products. Ideally, the recombinant protein products should be producedin an easily collectable tissue or biological fluid (e.g. hair, milk).Furthermore, expression in tissues that are targets of BP-1 action (e.g.bone, kidney) should be limited. In order to achieve these goals, theenhancer and promoter elements of the transcription unit are chosen sothat the BP-1 coding sequence is mainly expressed in the appropriatetissue (e.g. keratinocytes, mammary epithelium).

Fusion Protein in Bacteria

For some aspects of the present invention, it is desirable to produce afusion protein comprising a BP-1 polypeptide or at least one fragmentthereof or their derivatives and an amino acid sequence from anotherpeptide or protein, examples of the latter being enzymes such asbeta-galactosidase, phosphatase, urease and fusion proteinsincorporating purification moieties such as His-tags, FLAG-tags,myc-epitope tags and the like. Most fusion proteins are formed by theexpression of a recombinant gene in which two coding sequences have beenjoined together such that their reading frames are in phase.

For expression in E. coli, suitable expression vectors include pTRC(Amann et al. (1988) Gene 69: 301-315); pET-11d (Novagen, Madison,Wis.); pGEX (Amrad Corp., Melbourne, Australia); pMAL (N.E. Biolabs,Beverly, Mass.); pRIT5 (Pharmacia, Piscataway, N.J.); PSEM (Knapp et al.(1990) BioTechniques 8: 280-281); pQE30 (Qiagen, Germany); and pTrxFus(Invitrogen, Carlsbad, Calif.). The use of pTRC and pET-11d will lead tothe expression of unfused protein. The use of pGEX, pMAL, pRIT5, pSEM,pQE30, and pTrxFus will lead to the expression of BP-1 fused toglutathione S-transferase (pGEX), maltose E binding protein (pMAL),protein A (pRIT5), truncated β-galactosidase (PSEM), hexahistidine(His6), or thioredoxin. When a BP-1, fragment, or fragments thereof isexpressed as a fusion protein, it is particularly advantageous tointroduce an enzymatic cleavage site at the fusion junction between thecarrier protein and the BP-1 or fragment thereof. A BP-1 or fragmentthereof may then be recovered from the fusion protein through enzymaticcleavage at the enzymatic site and biochemical purification usingconventional techniques for purification of proteins and peptides.Suitable enzymatic cleavage sites include those for blood clottingFactor Xa or thrombin for which the appropriate enzymes and protocolsfor cleavage are commercially available from for example Sigma ChemicalCompany, St. Louis, Mo. and N.E. Biolabs, Beverly, Mass.

Host cells can be transformed to express the nucleic acid sequencesencoding the BP-1 of the invention using conventional techniques such ascalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, or electroporation. Suitable methodsfor transforming the host cells may be found in Sambrook et al. supra,and other laboratory textbooks. The nucleic acid sequences of theinvention may also be synthesized using standard techniques.

Chemical Synthesis

Homologues and derivatives of BP-1 is meant to include syntheticderivatives thereof. The nucleotide sequences encoding BP-1 can be usedto chemically synthesize the entire protein or generate any number offragments (peptides) by chemical synthesis by well known methods (e.g.,solid phase synthesis). All such chemically synthesized peptides areencompassed by the present invention. Alternatively, proteins orpeptides can be linked in vitro by chemical means. All such fusionprotein or hybrid genetic derivatives of BP-1 or its encoding nucleotidesequences are encompassed by the present invention. Accordingly, thepresent invention extends to isolated BP-1, fragments thereof and theirderivatives, homologues and immunological relatives made by recombinantmeans or by chemical synthesis.

vii) Methods of Purification of Recombinant BP-1 Protein Products.

BP-1 and fragments (peptides) thereof can be purified from cell culturemedium, host cells, or both using techniques known in the art forpurifying peptides and proteins, including ion-exchange chromatography,hydrophobic chromatography, gel filtration chromatography,ultrafiltration, electrophoresis and immunopurification with antibodiesspecific for BP-1 or fragments thereof. The terms isolated and purifiedare used interchangeably herein and refer to peptides, protein, proteinfragments, and nucleic acid sequences substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when synthesized chemically.

Typically, recombinant proteins found in culture medium or biologicalfluids are purified by successive steps of chromatography according tomethods known to an experimentator skilled in the art. According to apreferred embodiment of the invention, accumulation of secretedrecombinant BP-1 is done by incubating engineered mammalian cells inserum-free medium, in order to limit the amount of contaminatingproteins found in the medium. This medium is then diluted with columnbuffer (neutral HEPES-buffered saline) and passed on a cation-exchangecolumn (e.g. Sepharose™ SP, Amersham Pharmacia Biosciences). Since theisoelectric point of human BP-1 is 9.62, it binds strongly to thenegatively-charged resin in column buffer. After extensive washes withcolumn buffer, bound proteins are eluted with a salt gradient. Fractionsare collected and assayed for BP-1 immunoreactivity by ELISA. Underthese conditions, the BP-1 products elute at a salt concentration of250-300 mM. Further purification is achieved by affinity chromatography.Monoclonal or polyclonal antibodies specific to BP-1 (e.g. raisedagainst AG2, see section v) are covalently attached to a resin, eitherdirectly (e.g. CNBR-Sepharose™) or through binding and crosslinking toimmobilized protein A. In the latter case, protein A specifically bindsthe Fc portion of the antibodies, thereby avoidingcrosslinking/inactivation of their F(ab)₂ portion to the resin andincreasing the binding capacity of the affinity resin. BP-1-containingfractions from the ion-exchange column are pooled, diluted to a saltconcentration of approximately 100 mM and passed over the affinitycolumn at neutral pH. After washes, bound proteins are eluted underbasic (pH 10) and acidic (pH 2.9) conditions. Fractions are collected,neutralized and assayed for BP-1 immunoreactivity by ELISA.BP-1-containing fractions are pooled and, if necessary, dialyzed againstphysiological saline and concentrated (e.g. by lyophilization).

viii) Recombinant Adenoviruses Expressing BP-1 cDNA for Gene Delivery

Since processing of BP-1 to bioactive products may occur uniquely inosteoblasts or within the bone microenvironment, it may be advantageousto express a BP-1 coding sequence in cultured osteoblasts or directly invivo. Recombinant adenovirus particles are particularly useful todeliver a transcription unit into a whole organism or into cells thatare difficult to transfect by conventional methods such asdifferentiated osteoblasts (Ragot et al., 1998). Standard methodologymay be used to insert a transcription unit in an adenoviral genome andpackage the resulting recombinant adenoviral genome within an infectiousviral particle. Briefly, the transcription unit comprising a BP-1 codingsequence is cloned in a plasmid between regions of the adenovirusserotype 5 genome. This plasmid is transfected, along with areplication-defective viral genome, in a cell line that can complementthe replication defect, usually HEK293 cells. Homologous recombinationbetween a plasmid and a viral genome generates a recombinant viralgenome having inserted a BP-1 transcription unit. This recombinant viralgenome is subsequently packaged into infectious particles. Typically,the replication-defective viral genome minimally lacks the E1 proteinsuch that it can not replicate in infected cells unless these are HEK293cells that endogenously synthesize the E1 protein (Louis et al., 1997).The recombinant adenovirus can be propagated in HEK293 cells to veryhigh titers (>10¹⁰ pfu/ml). Stocks of recombinant adenoviral particlesare concentrated and purified by centrifugation on cesium chloridegradient according to standard procedures.

The resulting recombinant adenovirus can be used to infect large numbersof various cell types, including primary osteoblasts, to producerecombinant BP-1. It can also be used as a delivery system for systemicor local gene therapy or other protocols that may require overexpressionof BP-1 in vivo.

ix) Therapeutic Utility of BP-1 Products

Considering the fact that BP-1 is specifically expressed in theosteoblast lineage, BP-1 products may regulate bone cell proliferationand/or bone cell differentiation and/or of osteoblast activity, via anautocrine, paracrine or endocrine pathway. BP-1 products may alsocontrol osteoclast activity via a paracrine or endocrine pathway. Ineither cases, pharmaceutical compositions containing a therapeuticallyeffective amount of BP-1 products may be useful in the treatment of bonediseases, particularly in those characterized by bone loss such asosteoporosis. The BP-1 compositions may be further employed in methodsfor treating bone fractures or defects. Drugs that augment, mimick,antagonize or blunt the activity of BP-1 products may also be beneficialfor the treatment of bone diseases. Considering the in vitro effect ofour BP-1 preparations (see Example 4), drugs that antagonize or bluntthe activity of BP-1 products could be particularly useful to treatosteopenic or osteoporotic conditions.

Examples of drugs that augment the activity of BP-1 products includechemicals that activate proteins involved in the transcription of theBP-1 gene, thereby upregulating its expression. Drugs that inhibit thedegradation of a given BP-1 product should also lead to increasedactivity of this BP-1 product. Examples of drugs that mimick theactivity of BP-1 products include peptidomimetics or peptides, modifiedor not, corresponding to fragments of BP-1 that regulate bone or kidneyfunctions. In most cases, such drugs are agonists of the receptor thatbinds a given BP-1 product.

Examples of drugs that antagonize or blunt the activity of BP-1 productsinclude a) one or a mixture of antisense oligonucleotides blocking theexpression or translation of the BP-1 mRNA; b) one or a mixture ofantibodies raised against a given BP-1 product quenching the activity ofthis product; c) an antagonist binding the cognate receptor of a givenBP-1 product; d) a molecule inhibiting the enzymes responsible for theprocessing of the BP-1 precursor to a given product.

Phosphatonin is a putative hormone which regulates phosphate retentionby kidney tubules. It has been hypothesized that phosphatonin isproduced by cells of the osteoblast lineage and is a substrate for Phex,a metallopeptidase found at the cell surface of osteoblasts andosteocytes (Frota Ruchon et al., 2000; Ruchon et al., 1998; GenBank™ PIDaccession number g2499917). Our observations are consistent with thepossibility that a given BP-1 product could have phosphatonin activity.If this is the case, then hypophosphatemia could be remedied byinjection of a molecule that antagonize this activity. On the otherhand, hyperphosphatemia could be remedied by injection of a moleculethat stimulate or mimick phosphatonin activity. Alternatively,phosphatonin activity may be prolonged by using a drug that inhibits itsdegradation (e.g. inhibitors of the Phex endopeptidase activity). Suchproteolysis inhibitors could be useful to treat hyperphosphatemicconditions. Thus, drugs that augment, mimick, antagonize or blunt theactivity of BP-1 products may also be beneficial for the treatment ofdiseases characterized by abnormal serum phosphate levels.

Pharmaceutical Compositions

The present invention, therefore, provides a pharmaceutical compositioncomprising a therapeutically effective amount of BP-1 or derivatives,homologues or immunological relatives thereof and one or morepharmaceutically acceptable carriers and/or diluents. The activeingredients of a pharmaceutical composition comprising BP-1 iscontemplated to exhibit therapeutic activity when administered in amountwhich depends on the particular case. Dosage regime may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation. Theactive compound may be administered in a convenient manner such as bythe oral, intravenous (where water soluble), intramuscular,subcutaneous, intranasal, intradermal or suppository routes orimplanting (e.g., using slow release molecules). Depending on the routeof administration, the active ingredients which comprise thepharmaceutical composition of the invention may be required to be coatedin a material to protect the ingredients from the action of enzymes,acids and other natural conditions which may inactivate saidingredients.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders of the extemporaneous dispersion. In all cases the form must besterile and must be fluid to the extent that easy syringability exists.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of superfactants. The preventions of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When at least BP-1, or at least one fragment thereof is suitablyprotected as described above, the active compound may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with food of the diet. For oral therapeutic administration, theactive compound may be incorporated with excipients and used in the formof ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, becarried and may conveniently be between about 5 to 80% of the weight ofthe unit. The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(1) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment of bonedisease.

x) Screening Methods Using BP-1 Products

It is known in the prior art that proteins or peptides such as thosederived from BP-1 act through binding to cognate receptors. It ispossible to use labelled BP-1 products to identify such receptors. Thiscould be done as follows. BP-1 (PROT 1, SEQ ID NO: 20) can besynthesized in the presence of labeled amino acids (e.g. tritiatedL-leucine). The protein can be synthesized and labeled in vivo aftertransfection of a suitable host with a vector capable of expressing theBP-1 cDNA. Alternatively, transcripts can be synthesized, translated andlabeled in vitro starting with a plasmid harboring the BP-1 cDNAoperably linked to a DNA-dependent RNA polymerase promoter. Ideally, thein vitro translation step should be performed using rabbit reticulocytelysates containing microsomal membranes to allow for co-translationaland post-translational modifications to occur (Lingappa et al., 1979).In the case of peptides derived from BP-1 (PEPT 2A (SEQ ID NO: 22), PEPT3A (SEQ ID NO: 23), PEPT 2B (SEQ ID NO: 24), PEPT 3B (SEQ ID NO: 25)),these can be labeled during chemical synthesis (e.g. by incorporating abiotinylated derivative of amino acids) or by adding a N-terminaltyrosine residue that can be iodinated according to standard methods(Tsomides and Eisen, 1993). The labeled BP-1 product(s) can be purified(e.g. by affinity chromatography). To screen for cognate receptors, acDNA library is constructed according to standard protocols startingwith mRNA extracted from a target organ (e.g. bone, kidney). cDNAs arecloned in an expression vector downstream of strongly activeenhancer/promoter elements. The library of expression vectors isscreened for BP-1 product(s) binding activity as follows. The initialstep is to find a suitable host, i.e. one that does not bind the labeledBP-1 product(s) under basal conditions and in the absence of exogenousexpression vectors, preferably a mammalian cell line. Examples of hostsinclude, but are not limited to, HEK293 cells, CHO cells, COS cells(American Type Culture Collection no. CRL-1650) and CV-1 cells (AmericanType Culture Collection no. CCL-70). The library is then divided intopools (e.g. 500 clones per pool), each pool is transfected into the hostand incubated with the labeled BP-1 product(s). Expression vectors areextracted from cells transfected with a pool of clones that confersspecific binding of the labeled BP-1 product(s). Rounds of selection arerepeated until a single clone is identified. This clone encodes aputative receptor for BP-1 product(s). The receptor can be used toscreen for molecules that bind to it and for molecules that modulate thebinding of BP-1 product(s). Such assays can be performed on cellstransfected with a vector expressing the receptor cDNA.

xi) Diagnostic Assays

Based on the features described in the invention, it can be expectedthat methods to assay BP-1 products or nucleic acid sequences encodingBP-1 products will be useful as diagnostic reagents i.e to diagnose ormonitor diseases such as osteoporosis and disorders of phosphatemetabolism. Levels of BP-1 products or expression of the BP-1 gene maycorrelate with the occurrence of a bone disease. For example, the amountof BP-1 products may be elevated in bones of osteoporotic patients.Various immunological methods to assay soluble extracellular proteinsare well known in the prior art (e.g. radioimmunoassays, ELISA,‘sandwich’ ELISA). Such methods generally uses monoclonal or polyclonalantibodies specific to the protein of interest. In the case of thepresent invention, these antibodies could be raised against antigenicpeptides and purified as described in section v. Example 1 describes anenzyme-linked immunoadsorbent assay to quantify BP-1 products found incell culture medium. Other immunological methods to assay BP-1 productsin tissues or biological fluids may be developed by an experimentatorskilled in the art. Various hybridization-based methods to assay forspecific nucleic acids are well known in the prior art. In the case ofBP-1 for example, RNA extracted from cells of the bone marrow can belabeled during reverse transcription (e.g. radioactively orfluorescently) and single stranded cDNA can be hybridized to animmobilized nucleic acid probe comprising part of the BP-1 sequence(e.g. oligonucleotide, cDNA).

Proteins, peptides, or antibodies of the present invention can also beused for detecting and diagnosing bone or renal conditions that involvesecretion of BP-1. For example, diagnosis can be accomplished bycombining blood obtained from an individual to be tested with antibodiesto BP-1 and determining the extent to which antibody is bound to thesample.

Futhermore, it is expected that there are sequence polymorphisms in thenucleic acid sequence coding for BP-1, and it will be appreciated by oneskilled in the art that one or more nucleotides in the nucleic acidsequence coding for BP-1 may vary due to natural allelic variation. Anyand all such nucleotide variations and resulting amino acidpolymorphisms are within the scope of the invention. It may also beappreciated by one skilled in the art that BP-1 maybe a member of afamily of highly related genes. Nucleotide sequences and correspondingdeduced amino acid sequences of any and all such related family membersincluding BP-1 are within the scope of the invention.

EXAMPLES

As it will now be demonstrated by way of examples hereinafter, theinvention provides methods to produce, partially purify and characterizethe BP-1 protein products. Example 1 gives an example of a cell linesecreting BP-1 protein products in the culture medium. Example 2 givesan example of a method to partially purify the BP-1 protein productsfrom cell culture medium. Example 3 gives an example of recombinantadenovirus particles expressing a BP-1 coding sequence and uses thereof.Example 4 gives an example of treatment of primary cultures ofosteoblasts with medium containing BP-1 products.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described.

A) Materials and Methods

The following are experimental procedures and materials that were usedto describe the present invention.

Enzymes and Reagents

Restriction enzymes and DNA-modifying enzymes were purchased from NewEngland Biolabs (Cambridge, Ma.) unless otherwise stated. Syntheticoligonucleotides were obtained either from Hukabel Ltd. (Montreal,Quebec, Canada) or MWG Biotech Inc. (High Point, N.C.). Cell culturereagents were from Life Technologies (subsidiary of Invitrogen,Carlsbad, Calif.) unless otherwise stated. Chemicals were obtainedeither from Roche Molecular Biochemicals (Laval, Canada), Calbiochem(San Diego, Calif.) or Sigma (St. Louis, Mo.). Radiochemicals werepurchased from Amersham Pharmacia Biotech (Baie d'Urfé, Canada).Sequencing of DNA was done using the Dye terminator cycle sequencingDTCS™ sequencing kit (Beckman Coulter, Fullerton, Calif.) and a BeckmanCEQ 2000 automated sequencer.

RNA Extraction and Analysis

Total RNA was purified either by the guanidium isothiocyanate/acidphenol method (Chomczynski and Sacchi, 1987) or using the RNEASY™ kitaccording to the manufacturer's instructions (Qiagen, Mississauga,Canada). PolyA+ RNA was purified using the Oligotex™ kit according tothe manufacturer's instructions (Qiagen). Northern analysis wereperformed as follows. RNA was electrophoresed on 1.2% agarose/1.2%formaldehyde gels in 1×MOPS buffer (10 mM3-(N-morpholino)propanesulfonic acid, 4 mM sodium acetate, 0.5 mM EDTA,pH 7) and transferred onto a nylon membrane (Osmonics, Westborough, Ma.)by capillarity in 20×SSC. After UV crosslinking, the blot was incubatedin Church buffer (Church and Gilbert, 1984) for 4-8 hours at 63° C. andhybridized overnight in Church buffer containing approximately 1 ng/mlof ca155-cap1-c2-neg306 fragment that had been labelled with [α³²P]dCTPby random priming.

Cloning of Full Length Mouse cDNA Corresponding to ca155-cap1-c2-neg306

First, an expression vector was generated as follows. A 5498 bpSacII-ApaI fragment of pQBI25fc3 (qBiogene, Montreal, Canada) wasblunted and ligated unto itself to generate pCMVneo. Complementaryoligonucleotides 30-11 (SEQ ID NO. 35) and 30-12 (SEQ ID NO. 36) wereannealed and cloned in pCMVneo previously digested with BamHI and EcoRIand blunted using the Klenow fragment of DNA polymerase 1. The resultingplasmid is pCMVneoXN. The first strand of cDNAs was reverse transcribedat 42° C. from 5 μg of e15.5 mouse calvaria total RNA using a dT₁₅primer-linker (33-1V, SEQ ID NO: 37) and Superscript II™ (Invitrogen,Carlsbad, Calif.). After RNAse H treatment (Roche MolecularBiochemicals, Laval, Canada), 3/10 of the first strand cDNA preparationwas subjected to 25 cycles of PCR using DNA polymerases from the Titan™RT-PCR kit, forward primer 25-10G (25-10G,5′-aaacgctctgacftctcacaagatg-3′, SEQ ID NO. 38) and reverse primer18-30V (18-30V, 5′-gagatgaattcctcgagc-3′, SEQ ID NO. 39). Cyclingconditions were as follows: 94° C. for 45 seconds, 54° C. for 30seconds, 68° C. for 3 minutes. PCR products were cloned in pCMVneoXN togenerate GD25. Bacterial colonies were hybridized with aca155-cap1-c2-neg306 radioactive probe. Inserts from positive colonieswere sequenced.

Cloning of Vertebrate Homologs of BP-1

PolyA+ RNA from human bone marrow was purchased from Clontech Inc. (PaloAlto, Calif.). RT-PCR was performed on 50 ng of polyA+ RNA using forwardprimer 18-131G (SEQ ID NO. 40) and reverse primer 19-9G (SEQ ID NO. 41)and Titan™ one tube RT-PCR system according to the manufacturer'sinstructions (Roche Molecular Biochemicals). Cycling conditions (40cycles) were as follows: 94° C. for 1 minute, 54° C. for 1 minute, 68°C. for 1 minute. PCR products (predominant band at approximately 400 bp)were cloned in pBluescript 11 KS (Stratagene, La Jolla, Calif.) andinserts from randomly chosen colonies were sequenced.

Total RNA was extracted from UMR-106 cells (CRL-1661, American TypeCulture Collection, Manassas, Va.). This cell line is derived from a ratosteosarcoma and possesses characteristics of the osteoblast lineage(Partridge et al., 1983). The first strand of cDNAs was reversetranscribed at 42° C. from 5 μg of total RNA using 33-1V and SuperscriptII™ (Invitrogen). After RNAse H treatment (Roche Molecular Biochemicals)and purification using the minElute kit (Qiagen), ¼ of the first strandcDNA preparation was subjected to 35 cycles of PCR using the DNApolymerases from the Titan™ RT-PCR kit, forward primer 18-131G andreverse primer 18-30V. Cycling conditions were as follows: 94° C. for 1minute, 48° C. for 1 minute, 68° C. for 1.5 minute. RT-PCR products werepurified using the minElute kit. ⅙ of the initial PCR reaction wassubjected to 35 cycles of semi-nested PCR using the DNA polymerases fromthe Titan™ RT-PCR kit, forward primer 18-131G and reverse primer 21-9G(SEQ ID NO. 42). PCR products (predominant band at approximately 500 bp)were cloned in pBluescript II KS (Stratagene, La Jolla, Calif.) andinserts from randomly chosen colonies were sequenced.

Total RNA was extracted from the vertebrae and surrounding muscles of a2-month old birman python (˜130 g) using the TriZol™ reagent(Invitrogen). The first strand of cDNAs was synthesized at 42° C. for 1hour from 4 μg of total RNA using 0.5 μg of oligo-dT₁₈ and 200USuperscript II™ (Invitrogen) in a total volume of 20 μl. After RNAse Htreatment (2U added, Roche Molecular Biochemicals) and purificationusing the QIAQuick kit (Qiagen) with 30 μl of elution buffer, the firststrand cDNA preparation (2 μl) was subjected to 33 cycles of PCR usingTaq DNA polymerase, forward primer 23-4V (SEQ ID NO. 44) and reverseprimer 23-6V (SEQ ID NO. 45). Cycling conditions were as follows: 94° C.for 30 seconds, 56° C. for 30 seconds, 72° C. for 40 seconds. RT-PCRproducts were purified using the minElute kit. PCR products (predominantband at approximately 130 bp) were cloned in pBluescript II KS(Stratagene, La Jolla, Calif.) and inserts from randomly chosen colonieswere sequenced.

In Situ Hybridization

In situ hybridization on paraformaldehyde-fixed paraffin-embedded tissuesections was performed according to standard procedures (for example,see Wilkinson and Nieto, 1993). Briefly, [α³⁵S]UTP-labeled cRNA probewere synthesized in vitro from 0.5 μg of purified linearized templateDNA using DNA-dependent RNA polymerase (SP6, T7 or T3 RNA polymerase).After phenol/chloroform extraction and ethanol precipitation, quality ofthe probe was assessed by denaturing 4% polyacrylamide gelelectrophoresis. Tissue sections were deparaffinized, rehydrated,treated with 5 μg/ml of proteinase K (Roche Molecular Biochemicals) for15 minutes in 50 mM Tris-HCl pH 8/5 mM EDTA, refixed in 4%paraformaldehyde, acetylated with 0.25% (v/v) acetic anhydride in 0.1 Mtriethanolamine pH 8, dehydrated and air-dried. Prehybridization wascarried out for 2-4 hours at room temperature in 4×SET, 1× Denhardt'ssolution, 0.5 mg/ml ssDNA, 0.6 mg/ml yeast RNA, and 50% deionizedformamide. 1×SET is 0.15M NaCl, 0.03M Tris-HCl pH 8 and 2 mM EDTA. 1×Denhardt's solution is 0.02% (w/v) Ficoll, 0.02% (w/v)polyvinylpyrrolidone, 0.1% (w/v) BSA fraction V. ssDNA is salmon spermDNA that has been sonicated to obtain fragments averaging 2 kb, purifiedby phenol/chloroform extraction/ethanol precipitation/dialysis anddenatured by boiling. Hybridization was carried out for 12-16 hours at54° C. with 40,000 cpm/μl of CRNA probe in 4×SET, 1× Denhardt'ssolution, 0.1 mg/ml ssDNA, 0.1 mg/ml yeast RNA, 10% dextran sulfate, 10mM DTT, 0.1% SDS, and 50% deionized formamide. High stringency washeswere performed as follows: formamide wash for 30 minutes at 55° C. and30 minutes at 62° C.; 20 μg/ml RNAse A treatment in 3.5×SSC, 30 minutes,37° C.; formamide wash for 30 minutes at 62° C. Formamide wash is 50%deionized formamide/0.15M NaCl/0.15M sodium citrate/10 mM DTT. Afterrinses, slides were dehydrated, dipped in Kodak NTB-2 liquid emulsion(Intersciences, Markham, Canada), developed after 3-4 weeks ofexposition and counterstained by hematoxylin-eosin.

Cloning of Mouse BP-1 into a Mammalian Expression Vector

To optimize expression of BP-1 in heterologous systems, a vectorexpressing the coding sequence of mouse BP-1 was obtained as follows. A411 bp fragment was amplified from 10 ng of plasmid GD25 using forwardprimer 25-10G (SEQ ID NO: 38) and reverse primer 24-10G (SEQ ID NO: 43).The PCR reaction contained 25 pmoles/μl of each primer, deoxynucleotidesat 200 μM, dithiotreitol at 5 mM, the buffer and the enzyme mix from theTitan™ one-step RT-PCR kit (Roche Molecular Biochemicals). Cyclingconditions were as follows: 94° C. for 30 seconds, 56° C. for 30seconds, 68° C. for 30 seconds. The PCR product was cloned in the uniquePmel site of pCMVneoXN to generate GD23. The identity of the insert wasverified by DNA sequencing.

Production and Purifcation of Antisera

Peptides were synthesized, purified and coupled to activated keyholelympet hemocyanin via sulfhydryl groups. The peptide/carrier complex wasmixed with complete Freund's adjuvant and injected subcutaneously torabbits on day 1. On days 21, 35 and 49, the peptide/carrier complexmixed with incomplete Freund's adjuvant was again injected. Bloodsamples were collected on days 44 and 59 to determine the antiserumtiter by ELISA. Animals were exsanguinated on day 63. Immunoglobulinswere precipitated from pooled antisera and purified by peptide affinitychromatography according to standard protocols (Affinity Bioreagents,Golden, Co.).

Cell Culture and Transfection

HEK293A cells (purchased from Quantum Biotechnologies, Montreal, Canada)are grown in Dulbecco modified essential medium supplemented with 10%(v/v) fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin(referred hereafter as complete medium). Cells are passaged whenreaching 80-95% confluence by incubating with 0.05% (v/v) trypsin/0.5 mMEDTA (Wisent Inc., St-Bruno, Canada). Lipofection is performed asfollows. Lipid-DNA complexes (containing typically 1 μg of DNA) areformed using the Effectene™ reagent (Qiagen) according to themanufacturer's instructions. Cells are transfected the day after plating(typically 18,700 cells/cm²) by adding the lipid-DNA complex to theculture medium. After a 6 hour incubation, the medium is changed andcells are usually processed after 48 hours.

Immunofluorescence

All incubations and washes are done at room temperature. Cells arerinsed with PBS and fixed with 2% (w/v) paraformaldehyde in PBS. Cellsare washed with PBS. For detection of intracellular protein, cells arepermeabilized by incubation in 0.1% TRITON X-100™ in PBS for 4 minutesand washed twice with PBS. Cells are then incubated in 50 mM NH₄Cl inPBS for 10 minutes at room temperature and washed with PBS. Cells areincubated in PBS supplemented with 0.1% (w/v) bovine serum albuminfraction V and 2% (w/v) dried milk for 1 hour. Cells are then incubatedfor 1 hour with a 1/100 dilution (v/v) of antiserum raised against BP-1in PBS supplemented with 0.1% (w/v) bovine serum albumin and 0.5% (w/v)dried milk. Cells are washed twice with PBS and incubated for 1 hour in1/200 dilution of goat anti-mouse coupled to fluorescein isothiocyanate(Sigma) in PBS supplemented with 0.1% (w/v) bovine serum albumin. Cellsare washed twice and observed by fluorescence microscopy.

Western Analysis

Cell extracts. Cells are rinsed with PBS. Membrane proteins aresolubilized in 0.1 ml of Lysis buffer (50 mM Tris-Cl pH 8.0, 150 mMNaCl, 2 mM EDTA, 1% IGEPAL-630™ and 1% (v/v) protease inhibitor cocktail(Sigma)). Cell debris and insoluble material are pelleted bycentrifugation at 12,000 g for 5 minutes at 4° C. Protein concentrationin the supernatant is determined using the Bradford assay according tothe manufacturer's instructions (Bio-Rad, Hercules, Calif.).

Medium. 24 hours post-transfection, medium is replaced by mediumcontaining 0.5% (v/v) fetal bovine serum. The following day, this mediumis collected and centrifuged at 12,000 g for 5 minutes at 4° C. toremove cell debris. Proteins in the supernatant are precipitated byadding trichloroacetic acid at a final concentration of 10% (v/v) andincubating on ice for 1 hour. After centrifugation at 12,000 g for 15minutes at 4° C., the pellet is washed once with cold acetone, driedbriefly and resuspended in 24 μl of Lysis buffer.

Proteins are boiled for 5 minutes in the following Laemmli 1× solution:50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% sodium dodecylsulfate, 0.1% bromophenol blue, 10% glycerol. Proteins areelectrophoresed on denaturing polyacrylamide gel and transferred to 0.2μm nitrocellulose (Protran®, Schleicher&Schnell, Keene, N.H.) accordingto standard Western protocols. The nitrocellulose membrane is incubatedovernight in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 7.4, 137 mMNaCl, 2.7 mM KCl) supplemented with 5% (w/v) dried milk and 0.1% (v/v)TWEEN-20™. It is then incubated for 1 hour at room temperature with a1/800 dilution (v/v) of antiserum raised against BP-1 in TBSsupplemented with 2.5% (w/v) dried milk and 0.1% (v/v) TWEEN-20™. Themembrane is washed twice with TBS supplemented with 0.1% (v/v)TWEEN-20™. It is then incubated for 1 hour at room temperature with goatanti-mouse coupled to horseradish peroxidase (Sigma) diluted 1/30,000 inTBS supplemented with 2.5% (w/v) dried milk and 0.1% (v/v) TWEEN-20™.The membrane is washed twice with TBS supplemented with 0.1% (v/v)TWEEN-20™. Detection of the protein bound to the antibody complex isperformed with the ECL™ reagent according to the manufacturer'sinstructions (Amersham Pharmacia Biotech, Baie d'Urfé, Canada).

Primary Culture of Rat Calvarial Osteoblasts

Calvariae were dissected from embryonic rats from days 19-21 ofgestation. Osteoblasts were isolated by 5 successive digestions of thetissues with a mix of collagenase (2 mg/ml, Worthington, N.J.), trypsin(0.05% w/v, Roche Molecular Biochemicals, Ind.) and EDTA (0.02% w/v) for20 minutes with agitation at 37° C. Cells from digests 2 to 5 wereseeded at 10,000 cells/cm² and cultured in AMEM (Invitrogen, Carlsbad,Calif.) supplemented with 10% (v/v) FBS, 20 mM HEPES pH 7.4, 2 mML-glutamine, 5 mM β-glycerophosphate, 50 μg/ml ascorbic acid, 100U/mlpenicillin, 100 μg/ml streptomycin. Medium was changed every 2-3 days.

Incorporation of ⁴⁵Ca into Primary Cultures of Rat Calvarial Osteoblasts

Cells were incubated in medium containing 1 μCi/ml ⁴⁵CaCl₂ to assaycalcium uptake into mineralized matrix. Medium was collected 48 hourslater from each well and added to 4 ml of scintillation cocktail, andcounted on a scintillation counter. The cells were washed twice in PBSthen scraped into 5001 μl of lysis buffer (20 mM Tris, 0.5 mM MgCl₂,0.1% (v/v) Triton X-100™). Cellular debris was spun out, the lysisbuffer removed and stored and the pellet resuspended in 1 ml 0.5M EDTA.The pellet in EDTA was placed overnight on a rocking platform then thedebris spun out and the EDTA solution removed and stored. The pellet wasthen resuspended in 1 ml 0.5M NaOH and placed overnight on a rockingplatform. The remaining cell debris was spun out and the NaOH removedand stored. To ascertain ⁴⁵Ca incorporation, 100 μl of the lysis bufferand 500 μl of the EDTA and NaOH fractions were counted in 4 ml ofscintillation fluid. The proportion of ⁴⁵Ca taken up into the cells ineach well was then calculated as a percentage of the total counts ineach well.

B) Example 1 Cell Lines Secreting BP-1 Protein Products in the CultureMedium

The expression vector used to obtain stable cell lines producing BP-1 isschematized on FIG. 7A. Plasmid RC33 (see provisional patent applicationand international patent application PCT/CA02/00997) was digested withKpnI and EcoRI and plasmid GD23 (see Materials and Methods) was digestedwith KpnI and Mlul. Both digests were treated using the Klenow fragmentof DNA polymerase I to blunt the 5′ protruding extremities generated byEcoRI and Mlul. The 5194 bp fragment of the RC33 digest and the 473 bpfragment of the GD23 digest were purified and ligated. The resultingvector is designated GD46. A similar vector comprising the codingsequence for human BP-1 was obtained and is designated GD45. One of thetranscription unit of GD46 comprises the cytomegalovirus immediate earlygene enhancer/promoter regions (700) followed by the coding sequence formouse BP-1 (701) and a bovine growth hormone polyadenylation signal(702). GD46 also comprises a transcription unit to confer resistance topuromycin (703).

To obtain cell lines having integrated one or more copies of GD46 in itsgenome, 3.5 million of HEK293 cells were electroporated at 600V/cm witha mixture of 3.5 μg of ScaI-linearized expression vector and 15 μg ofdenatured salmon sperm DNA. Cells were plated and selection (2.5 μg/mlpuromycin) was applied 24 hours later. The concentration of puromycinwas reduced to 0.2 μg/ml on day 4 and colonies were picked on day 11.Clones were grown until confluence in 24-well plates. At this time,cells were incubated in 0.5 ml of medium without serum for 48 hours.One-fifth (100 μl) was analyzed by ELISA as follows. One volume (100 μl)of 2× carbonate buffer (13 mM Na₂CO₃/35 mM NaHCO₃) was added to thesample. Samples were transferred in wells of a high-binding capacitypolystyrene plate (Corning, USA) and incubated 1 hour at 37° C. Themedium was then aspirated and the well was washed once with TBST(Tris-buffered saline pH 7.4 [TBS; 100 mM Tris], 0.1% (v/v) Tween-20™).Non-specific binding sites were blocked for 1 hour at 37° C. with 300 μlof TBS supplemented with 5% (w/v) of non fat milk. After 5 washes withTBST, incubation proceeded for 1 hour at 37° C. with 200 μl of a 1/2500dilution of the affinity-purified antibody against the C-terminalportion of BP-1 in TBST supplemented with 2.5% (w/v) of non fat milk.The plate was then washed 5 times with TBST. Samples were incubated for1 hour at 37° C. with 200 μl of a 1/30,000 dilution of anti-rabbit IgGcoupled to horseradish peroxydase (Sigma, St. Louis, Mo.). After 5washes with TBST, the antibody complex was revealed with Sigma Fast™o-phenylenediamine dihydrochloride according to the manufacturer'srecommendations. Reactions were stopped by adding 25 μl of 5N sulfuricacid per well. Absorbance at 490 nm was determined using a plate reader.Known quantities of antigenic peptide are also assayed to determine astandard curve (FIG. 7B). The clones of GD46 transfectants secretingimmunoreactive BP-1 products in the culture medium were expanded. Theamount of BP-1 products released by each clone was calculated byregression analysis after assaying varing volumes of culture medium. Thehighest expressor (clone 293-GD46-7) was found to secrete 8 pg of BP-1products per cell in the culture medium in 48 hours. This corresponds toa production of approximately 10 mg/l for a confluent monolayer in a 175cm² flask. We also obtained a cell line overexpressing the human BP-1coding sequence (293-GD45-58B). Confluent 175 cm² monolayers of thesecells secrete approximately 0.6 mg of BP-1 products per liter of culturemedium in 48 hours.

To ascertain that the cell lines we derived were of monoclonal originand would not be diluted by non BP-1-expressing cells upon passaging,immunofluorescence analysis was performed on confluent monolayers of293A-GD46-7 using an antibody directed against the C terminal portion ofBP-1. BP-1 immunoreactivity was seen in the secretory apparatus in over98% of the cells.

C) Example 2 Method to Partially Purifly the BP-1 Protein Products fromCell Culture Medium

Six million cells stably overexpressing mouse BP-1 (clone 293-GD46-7)were seeded in a 175 cm² flask and grown for 48 hours in DMEMsupplemented with 10% FBS, at which stage the monolayer wasapproximately 90% confluent. The cells were then washed 2 times withpre-warmed phosphate buffered saline and once with serum-free DMEM. Thecells were incubated for 48 hours in 20 ml of serum-free DMEM. Themedium was then collected and spun down at 800 g for 2 minutes to removefloating cells and debris. All subsequent procedures were performed at4° C. using a BioRad BioLogic™ LP Chromatography system. The conditionedmedium containing BP-1 products was first diluted 2-fold withequilibration buffer (Buffer A: 25 mM HEPES pH 7.8 and 100 mM NaCl) andfiltered through a 0.45 μm Filtropur™ S membrane. The diluted medium wasthen loaded at a flow rate of 2 ml/min on a column (1.5 cm×20 cm) packedwith 3.5 ml gel bed of a Sepharose™ SP cation exchange resin(Amersham-Pharmacia Biosciences). The flow-through was collected untilthe absorbance at 280 nm returned to baseline. The resin was washed with5 bed volume of Buffer A at 2 ml/min. The bound-proteins were eluted at1 ml/min with 50 ml of a linear gradient from 0-100% of Buffer B (25 mMHEPES pH 7.8 and 1 M NaCl). Fractions of 1 ml were collected andmonitored for the presence of BP-1 by ELISA (see Example 1). Thefractions containing BP-1 were pooled and concentrated 10-fold on anAmicon Centriplus™ YM-3 (3 kDa cut-off membrane) by centrifugation at3400 g at 4° C. No significant loss of BP-1 immunoreactivity occurredduring this concentration procedure.

FIG. 8 shows the results of the initial steps of the purification schemedescribed above. The fractions collected from the cation-exchange columnwere individually assayed for BP-1 immunoreactivity by ELISA (FIG. 8A).The input (conditioned medium 0.9% of total), the flow through (0.9% oftotal) and BP-1-containing fractions (0.8% of total) were analyzed bySDS-PAGE 10-20% (FIG. 8B, lanes 810, 811, 812, respectively). Afterelectrophoresis, the gel was stained by PlusOne™ silver staining kit(Amersham Pharmacia Biosciences). Lane 800 shows the migration ofmolecular weight markers. Arrow 813 points to a BP-1 product. Arrowhead814 points to aprotinin, a contaminating protease inhibitor included inthe conditioned medium before the chromatographic step. A duplicate gelwas transferred onto nitrocellulose membrane and BP-1 products wererevealed by immunostaining (FIG. 8C, lane 820:input, lane 821:flowthrough, lane 822: BP-1-containing fractions). Results indicate that thebulk of the proteins in the culture medium do not bind the cationexchange column (compare lanes 810 and 811). By Coomassie and silverstaining, we estimate that these initial steps of purification allowed a25-fold enrichment in BP-1 products. As can be seen on the Sepharose™ SPelution profile (FIG. 8A), BP-1 products eluted at a salt concentrationof approximately 300 mM NaCl. Importantly, the immunoreactive fractionscontain products of lower apparent molecular weight (lane 822, band at˜6 kD) in addition to the ˜13 kD form of BP-1. This indicates that thepartial purification scheme described above can enrich for all BP-1products secreted by an overexpressing cell line.

D) Example 3 Recombinant Adenovirus Particles Expressing the BP-1 CodingSequence and Uses Thereof

This example illustrates the various functionalities of anadenovirus-based expression vector designed according to the presentinvention. To obtain a ‘shuttle’ vector for inserting a transcriptionunit into an adenoviral genome, a 1689 bp Stul-Nael fragment from GD23(see Materials and Methods) was blunted and cloned in the EcoRV site ofpQBI-AdBN (qBiogene, Montreal, Canada) which had been previouslyengineered to replace the unique ClaI site with a unique Pmel site. Theresulting vector is designated GD28b and comprises a transcription unitfor mouse BP-1 coding sequence (901) flanked by nucleotides 1-102 (902)and nucleotides 3334-5779 (903) of the Adenovirus serotype 5 genome(GenBank™ accession number 9626187). A map of GD28b is given in FIG. 9A.

The transcription unit for BP-1 comprised in GD28b was incorporated inan adenoviral genome by in vivo homologous recombination. This was doneby co-transfecting 5 μg of Pmel-linearized GD28b with 5 μg ofAdCMVIacZΔE1/ΔE3, a replication-defective genome obtained commercially(qBiogene, Montreal, Canada). Co-transfection of DNA molecules in HEK293cells was carried out by means of a calcium phosphate precipitate usingstandard protocols. Two days post-transfection, cells were overlaid withmedium containing 1.25% (w/v) low melting agarose. Recombinant viralgenome resulting from homologous recombination between GD28b and thereplication-defective adenoviral genome can be propagated in HEK293cells, as indicated by the appearance of viral plaques starting at day 8post-infection. Plaques were picked at day 14 post-transfection. Afterelution, viral particles ( 1/10 of the elutate) were used to infect3×10⁵ HEK293 cells to assay for BP-1 expression. This was done bymeasuring BP-1 immunoreactivity by ELISA in 1/10 of the culture medium 2days post-infection. Expression of BP-1 was also confirmed by Northernand Western analysis. A stock of recombinant adenoviral particles(Ad5GD28b) was obtained after 2 successive rounds of plaque-purificationaccording to standard protocols. This stock was amplified on HEK293cells. At the time of full cytophathic effect, the infected cells wereharvested and the viral particles were purified on a discontinuouscesium chloride gradient and on a continuous cesium chloride gradientaccording to standard protocols (O.D.260 Inc., Boise, Id., USA).

The following experiment was performed to assess the efficiency of BP-1gene transfer in osteoblasts by recombinant adenoviruses. Primarycultures of osteoblasts were grown in vitro (see Materials and Methods).Cells reached confluence 5 days after seeding, at which time theystarted depositing an extracellular collagen matrix. Mineralization ofthis matrix was routinely visible around days 13-15. At day 11, cellswere infected with CsCl-purifled Ad5GD28b at a multiplicity of infectionof 100 pfu/cell for 3 hours. Relative levels of BP-1 products weredetermined by ELISA on aliquots ( 1/40) of culture medium taken 2, 4, 7and 10 days post-infection (FIG. 9B). Results show that BP-1immunoreactivity is detected as early as 2 days post-infection, peaks atday 4 and is still detectable 10 days post-infection. These resultsindicate that a preparation of Ad5GD28b can be used to express highlevels of BP-1 in osteoblasts.

E) Example 4 Treatment of Osteoblasts with Medium Containing BP-1Products

This example demonstrates the effects of treating primary cultures ofosteoblasts with medium containing BP-1 products. The BP-1-containingmedium was obtained by transient transfection of 1.5 million HEK293Acells with 2 μg of GD23, an expression vector for mouse BP-1 cDNA.Control medium was obtained by similarly transfecting pCMVneo, a controlexpression vector. At 24 hours post-transfection, cells were washedtwice with serum-free αMEM. The transfected cells were then incubatedfor 48 hours in primary culture medium. BP-1 products were characterizedby Western analysis using an antiserum raised against a C-terminalportion of BP-1 (SEQ AG3). The concentration of BP-1 products wasestimated at 5-10 μg/ml with the bulk of immunoreactive protein havingan apparent molecular weight of ˜13 kD and aminor fraction having anapparent molecular weight of ˜6 kD after SDS-PAGE (e.g. FIG. 6B).

Primary cultures of osteoblasts prepared as described in Materials andMethods were cultivated in a ⅙ (v/v) dilution of either BP-1-containingor control medium. β-glycerophosphate (10 mM) was added to the culturemedium from day 12. The effect of the treatment was determined bymeasuring osteocalcin expression and matrix mineralization, both markersof the mature osteoblastic phenotype. As shown in FIG. 10, treatmentwith BP-1-containing medium (1002) for 18 days completely suppressedosteocalcin (OCN) expression compared to treatment of cells with controlmedium (1001). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)expression in the two cell populations was similar, demonstrating nogeneral effects on the cell populations. Incorporation of ⁴⁵Ca was alsomarkedly reduced in the cells treated with BP-1-containing mediumcompared to cells treated with control medium (−60%, p<0.01). Theseresults suggest that BP-1 products restrict the progression from earlyto late stage osteoblasts.

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1. An isolated polypeptide comprising SEQ ID NO: 9, wherein thepolypeptide regulates at least one of bone cell proliferation, bone celldifferentiation and osteoblast activity.
 2. An isolated fragment of thepolypeptide of claim 1 comprising SEQ ID NO: 20, wherein the fragmentregulates at least one of bone cell proliferation, bone celldifferentiation and osteoblast activity.
 3. The fragment of claim 2further comprising a label selected from the group consisting of one ormore iodinated amino acid residues, one or more biotinylated amino acidresidues, and one or more tritiated L-leucines.
 4. A pharmaceuticalcomposition comprising the fragment of claim 2 and a pharmaceuticallyacceptable carrier.
 5. An isolated fragment of the polypeptide of claim1 comprising SEQ ID NO: 46, wherein the fragment regulates at least oneof bone cell proliferation, bone cell differentiation and osteoblastactivity.
 6. The fragment of claim 5 further comprising a label selectedfrom the group consisting of one or more iodinated amino acid residues,one or more biotinylated amino acid residues, and one or more tritiatedL-leucines.
 7. A pharmaceutical composition comprising the fragment ofclaim 5 and a pharmaceutically acceptable carrier.
 8. An isolatedfragment of the polypeptide of claim 1 comprising SEQ ID NO: 21, whereinthe fragment regulates at least one of bone cell proliferation, bonecell differentiation and osteoblast activity.
 9. The fragment of claim 8further comprising a label selected from the group consisting of one ormore iodinated amino acid residues, one or more biotinylated amino acidresidues, and one or more tritiated L-leucines.
 10. A pharmaceuticalcomposition comprising the fragment of claim 8 and a pharmaceuticallyacceptable carrier.
 11. An isolated fragment of the polypeptide of claim1 comprising SEQ ID NO: 47, wherein the fragment regulates at least oneof bone cell proliferation, bone cell differentiation and osteoblastactivity.
 12. The fragment of claim 11 further comprising a labelselected from the group consisting of one or more iodinated amino acidresidues, one or more biotinylated amino acid residues, and one or moretritiated L-leucines.
 13. A pharmaceutical composition comprising thefragment of claim 11 and a pharmaceutically acceptable carrier.
 14. Anisolated fragment of the polypeptide of claim 1 consisting of SEQ ID NO:26.
 15. The fragment of claim 14 further comprising a label selectedfrom the group consisting of one or more iodinated amino acid residues,one or more biotinylated amino acid residues, and one or more tritiatedL-leucines.
 16. A pharmaceutical composition comprising the fragment ofclaim 14 and a pharmaceutically acceptable carrier.
 17. An isolatedfragment of the polypeptide of claim 1 consisting of SEQ ID NO:
 27. 18.The fragment of claim 17 further comprising a label selected from thegroup consisting of one or more iodinated amino acid residues, one ormore biotinylated amino acid residues, and one or more tritiatedL-leucines.
 19. A pharmaceutical composition comprising the fragment ofclaim 17 and a pharmaceutically acceptable carrier.
 20. An isolatedfragment of the polypeptide of claim 1 consisting of SEQ ID NO:
 28. 21.The fragment of claim 20 further comprising a label selected from thegroup consisting of an iodinated tyrosine residue at the fragment'sN-terminal, a biotinylated derivative of an amino acid, and a tritiatedL-leucine.
 22. A pharmaceutical composition comprising the fragment ofclaim 20 and a pharmaceutically acceptable carrier.
 23. The polypeptideof claim 1 further comprising a label selected from the group consistingof one or more iodinated amino acid residues, one or more biotinylatedamino acid residues, and one or more tritiated L-leucines.
 24. Apharmaceutical composition comprising the polypeptide of claim 1 and apharmaceutically acceptable carrier.
 25. The polypeptide of claim 1,consisting of SEQ ID NO:
 9. 26. An isolated fragment of the polypeptideof claim 1, consisting of SEQ ID NO:
 20. 27. An isolated fragment of thepolypeptide of claim 1, consisting of SEQ ID NO:
 21. 28. An isolatedfragment of the polypeptide of claim 1, consisting of SEQ ID NO:
 46. 29.An isolated fragment of the polypeptide of claim 1, consisting of SEQ IDNO:
 47. 30. An isolated polypeptide encoded by the nucleotide sequenceof SEQ ID NO: 2.